PIT-1 ACTIVATION OF THE GROWTH HORMONE CHROMATIN LOCUS

描述(由申请人提供) 这项申请的目的是大幅拓宽和加强对应聘者在分子生物学领域的培训。这将在一个明确的指导计划下完成，方法是扩大正在进行的小鼠远程基因激活转基因研究(AIM I)的范围，并通过获得一套全新的生物化学和结构生物学工具来制定和执行一套补充的体外研究(AIM II)。提出的实验重点是激活人类生长激素(HGH)染色质基因座。生长激素基因已经成为研究组织特异性和发育调控基因表达的范例。HGH基因簇特别有用，因为它还可以作为远程基因激活的信息模型。一组远端调控元件，包括一个位点控制区(LCR)，对hGH基因簇在脑下垂体和胎盘中的表达起着重要的调控作用。HGH LCR决定簇在很长的距离上以发育有序和组织特异性的方式激活它们的目标基因。深入了解hGH LCR的结构、功能和作用机制，对于理解hGH基因调控和扩展现有的基因调控机制模型具有重要意义。一套具体的科学目标将在指导环境中得到解决，这将扩大候选人的专业能力和科学技能。目的通过建立一组新的转基因小鼠品系来扩展hGH LCR活性的表征，以评估hGH基因一旦被激活是否需要hGH基因HSI上的决定因素来维持转录活性状态，并确定HSI上的Pit-1结合位点是否能够自主靶向组蛋白乙酰化。AIM II的体外研究将测试纯化的重组Pit-1与重组核小体中LCR的Pit-1元件结合的能力，随后将CBP/p300组蛋白乙酰化因子招募到Pit-1：核小体复合体中，并激活来自重组染色质模板的转录。这些目标将在两个人的指导下实现，这些人致力于候选人的培训和各自领域的专家。申请的最终目标是促进对基因调控的理解，并使候选人能够承担学术机构的独立研究项目。

DESCRIPTION (provided by applicant) The objective of this application is to substantially broaden and strengthen the training of the candidate in the field of molecular biology. This will be accomplished under a defined mentoring program by extending the scope of ongoing mouse transgenic studies of long-range gene activation (Aim I) and by acquiring an entirely new set of biochemistry and structural biology tools required to formulate and execute a complementary set of investigations in vitro (Aim II). The experiments that are proposed focus on activation of the human Growth Hormone (hGH) chromatin locus. The GH gene has served as a paradigm for the study of tissue-specific and developmentally-regulated gene expression. The hGH gene cluster is of particular utility as it also serves as an informative model for long-range gene activation. A set of distal regulatory elements, comprising a locus control region (LCR), exerts major regulatory control over expression of the hGH gene cluster in both the pituitary and placenta. HGH LCR determinants activate their target genes over long distances and in a developmentally ordered and tissue-specific manner. A detailed understanding of hGH LCR structure, function, and mechanisms of action is of central importance to understanding of hGH gene regulation and to expanding the available mechanistic models of gene control. A set of specific scientific goals will be addressed within a mentoring environment that will expand the candidate's professional abilities and scientific skills. Aim I will expand ongoing characterization of hGH LCR activities by developing a novel set of transgenic mouse lines to assess whether determinants at HSI of the LCR are required to maintain the hGH gene in a transcriptionally active state once it is activated, and to determine whether the Pit-1 binding sites at HSI can autonomously target histone acetylation. The in vitro studies of Aim II will test the ability of purified recombinant Pit-1 to bind to Pit-1 elements of the LCR in reconstituted nucleosomes, to subsequently recruit CBP/p300 histone acetylation factors to the Pit-1: nucleosome complexes, and to activate transcription from a reconstituted chromatin template. These Aims will be carried out under the mentorship of two individuals dedicated to the candidate's training and expert in their respective fields. The ultimate goal of the application is to advance the understanding of gene regulation and to position the candidate to assume an independent research program at an academic institution.